The vitamin K-dependent coagulation Factor IX/IXa has an important role in hemostasis and can contribute to the pathogenesis of thrombosis (1-4). In view of the well-known rapid clearance of Factor IX from the intravascular space, the association of infused and endogenous Factor IX with the vessel wall (5-6), and in vitro studies demonstrating Factor IX binding to endothelium and platelets (7-9), studies have been performed to characterize the molecular basis of this coagulation protein-cell surface interaction.
At the level of the ligand, the amino terminal gamma-carboxyglutamic acid-containing domain of Factor IX has been shown to be essential for cell surface binding (10-14). At the level of the cell surface site, previous studies have suggested that Factor IX binding involves a protease sensitive polypeptide, which on endothelial cells (ECs) had a predicted Mr of .apprxeq.150 kDa (15), and on platelets appeared to involve proteins with Mr's of .apprxeq.150 kDa and .apprxeq.250 kDa (16).
Abbreviations: EC=endothelial cell; GST=glutathione S-transferase; PVDF=polyvinylidene difluoride; PVC=polyvinlychloride plate binding assay; tPA=tissue plasminogen activator; PAI-1=plasminogen activator inhibitor-1; IC=intracoronary.
To further characterize polypeptides which interact with Factor IX, we employed bovine lung extract as a starting material and purified the major species which binds Factor IX. A .apprxeq.55 kDa Factor IX binding polypeptide was isolated which also interacted similarly with Factor X and prothrombin. This polypeptide proved to be identical to calreticulin, a previously described intracellular calcium binding. Interaction of calreticulin/.apprxeq.55 kDa with these vitamin K-dependent coagulation factors was localized to the C-domain, but did not change their coagulant properties. In contrast, calreticulin bound to ECs in vitro, was associated with the vessel wall after intravenous infusion, and stimulated production of nitric oxide. Intracoronary administration of calreticulin prevented coronary thrombosis in a canine model without altering plasma clotting times or increasing extravascular bleeding. These results suggest that calreticulin has a novel, potentially useful antithrombotic function.